Computer-aided nucleic acid sequencing

• US 6,361,937 B1
• Filed: 03/19/1996
• Issued: 03/26/2002
• Est. Priority Date: 03/19/1996
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Stryer, Lubert
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/618,834
• Time in Patent Office: 2,198 Days
• Field of Search: 435/6, 536/24.3, 364/496, 364/497, 436/94, 935/76, 935/77, 702/19, 702/20, 422/68.1
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

A computer system for sequencing nucleic acids is provided. The computer system may be used for de novo sequencing of a nucleic acid sequence by analyzing the fluorescence intensities of hybridized nucleic acid probes on biological chips. The probes with the highest intensities are utilized to sequence the nucleic acid and related probes are analyzed to increase the accuracy of nucleic acid sequencing. The sequence of the nucleic acid sequence may be determined from hybridization intensities that do not allow identification of perfectly complementary probes.

Claims

1. A computer implemented method of sequencing a nucleic acid sequence, the method comprising the steps of:

inputting hybridization intensities into a computer for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence under conditions that do not allow identification of all nucleic acid probes of the plurality of nucleic acid probes that are perfectly complementary to part of the nucleic acid sequence;

the computer selecting nucleic acid probes from the plurality of nucleic acid probes, wherein the step of selecting nucleic acid probes includes selecting nucleic acid probes with highest numbers of single base mismatch neighbors among the probes, a single base mismatch neighbor being another probe that has the same sequence except for a single base that is different; and

the computer sequencing the nucleic acid sequence according to the selected nucleic acid probes.

2. A computer implemented method of sequencing a nucleic acid sequence, the method comprising the steps of:

inputting hybridization intensities into a computer for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence under conditions that do not allow identification of all nucleic acid probes of the plurality of nucleic acid probes that are perfectly complementary to part of the nucleic acid sequence;

the computer selecting nucleic acid probes from the plurality of nucleic acid probes, wherein the step of selecting nucleic acid probes includes selecting nucleic acid probes that have fewer than a predetermined number of base mismatches with another probe; and

the computer sequencing the nucleic acid sequence according to the selected nucleic acid probes.

3. A computer implemented method of sequencing a nucleic acid sequence, the method comprising the steps of:

inputting hybridization intensities into a computer for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the-nucleic acid sequence under conditions that do not allow identification of all nucleic acid probes of the plurality of nucleic acid probes that are perfectly complementary to part of the nucleic acid sequence;

the computer selecting nucleic acid probes from the plurality of nucleic acid probes, wherein the step of selecting nucleic acid probes includes selecting nucleic acid probes with highest hybridization intensities and the nucleic acid probes with the highest hybridization intensities are identified as having hybridization intensities above an intensity threshold; and

the computer sequencing the nucleic acid sequence according to the selected nucleic acid probes.

4. A computer implemented method of sequencing a nucleic acid sequence, the method comprising the steps of:

inputting hybridization intensities into a computer for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence under conditions that do not allow identification of all nucleic acid probes of the plurality of nucleic acid probes that are perfectly complementary to part of the nucleic acid sequence;

the computer selecting nucleic acid probes from the plurality of nucleic acid probes; and

the computer sequencing the nucleic acid sequence according to the selected nucleic acid probes, wherein the sequencing step includes the steps of;

aligning the selected nucleic acid probes so that bases that are common overlap; and

sequencing the nucleic acid sequence according to bases that occur most often at each position in the aligned nucleic acid probes.

5. The method of claim 4, wherein the nucleic acid sequence is sequenced as complementary to the bases that occur most often.

6. A computer implemented method of sequencing a nucleic acid sequence, the method comprising the steps of:

inputting hybridization intensities into a computer for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence;

the computer selecting nucleic acid probes with highest numbers of single base mismatch neighbors among the probes, a single base mismatch neighbor being another probe that has the same sequence except for a single base that is different; and

the computer sequencing the nucleic acid sequence according to the selected nucleic acid probes.

7. The method of claim 6, further comprising the step of selecting nucleic acid probes with highest hybridization intensities.

8. The method of claim 7, wherein the nucleic acid probes with the highest hybridization intensities are identified as having hybridization intensities above an intensity threshold.

9. The method of claim 6, wherein the step of selecting includes the step of calculating a number of single base mismatch neighbors for each nucleic acid probe.

10. The method of claim 6, wherein the step of selecting includes the step of comparing double base mismatch neighbors, a double mismatch neighbor being another probe that has the same sequence except for two bases that are different.

11. The method of claim 6, wherein the sequencing step includes the steps of:

aligning the selected nucleic acid probes so that bases that are common overlap; and

sequencing the nucleic acid sequence according to bases that occur most often at each position in the aligned nucleic acid probes.

12. The method of claim 11, wherein the nucleic acid sequence is sequenced as complementary to the bases that occur most often.

13. A computer program product that sequences a nucleic acid sequence, comprising:

code that receives as input hybridization intensities for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence;

code that selects nucleic acid probes with highest numbers of single base mismatch neighbors among the probes, a single base mismatch neighbor being another probe that has the same sequence except for a single base that is different;

code that sequences the nucleic acid sequence according to the selected nucleic acid probes; and

a computer readable storage medium that stores the codes.

14. The computer program product of claim 13, wherein the computer readable storage medium is a floppy drive, hard drive, CD-ROM, memory, system memory, memory cache, flash memory, or tape.

15. The computer program product of claim 13, further comprising code that selects nucleic acid probes with highest hybridization intensities.

16. The computer program product of claim 15, wherein the nucleic acid probes with the highest hybridization intensities are identified as having hybridization intensities above an intensity threshold.

17. The computer program product of claim 13, wherein the code that selects includes code that calculates a number of single base mismatch neighbor for each nucleic acid probe.

18. The computer program product of claim 13, wherein the code that selects includes code that compares double base mismatch neighbors, a double mismatch neighbor being another probe that has the same sequence except for two bases that are different.

19. The computer program product of claim 13, wherein the code that sequences includes:

code that aligns the selected nucleic acid probes so that bases that are common overlap; and

code that sequences the nucleic acid sequence according to bases that occur most often at each position in the aligned nucleic acid probes.

20. The computer program product of claim 19, wherein the nucleic acid sequence is sequenced as complementary to the bases that occur most often.

21. A computer implemented method of sequencing a nucleic acid sequence, the method comprising the steps of:

inputting hybridization intensities into a computer for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence;

the computer selecting nucleic acid probes that have fewer than a predetermined number of base mismatches with another probe; and

the computer sequencing the nucleic acid sequence according to the selected nucleic acid probes.

22. The method of claim 21, further comprising the step of selecting nucleic acid probes with highest hybridization intensities.

23. The method of claim 22, wherein the nucleic acid probes with the highest hybridization intensities are identified as having hybridization intensities above an intensity threshold.

24. The method of claim 21, wherein each nucleic acid probe includes n bases.

25. The method of claim 24, further comprising the steps of:

comparing bases at positions 1 through n of a first nucleic acid probe to bases at positions 1 through n of a second nucleic acid probe; and

counting base mismatches between the first and second nucleic acid probe.

26. The method of claim 24, further comprising the steps of:

comparing bases at positions 1 through n−

1 of a first nucleic acid probe to bases at positions 2 through n of a second nucleic acid probe; and

counting base mismatches between the first and second nucleic acid probe.

27. The method of claim 21, wherein the sequencing step includes the steps of:

aligning the selected nucleic acid probes so that bases that are common overlap; and

sequencing the nucleic acid sequence according to bases that occur most often at each position in the aligned nucleic acid probes.

28. The method of claim 27, wherein the nucleic acid sequence is sequenced as complementary to the bases that occur most often.

29. A computer program product that sequences a nucleic acid sequence, comprising:

code that receives as input hybridization intensities for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence;

code that selects nucleic acid probes that have fewer than a predetermined number of base mismatches with another probe;

code that sequences the nucleic acid sequence according to the selected nucleic acid probes; and

a computer readable storage medium that stores the codes.

30. The computer program product of claim 29, wherein the computer readable storage medium is a floppy drive, hard drive, CD-ROM, memory, system memory memory cache, flash memory, or tape.

31. The computer program product of claim 29, further comprising code that selects nucleic acid probes with highest hybridization intensities.

32. The computer program product of claim 31, wherein the nucleic acid probes with the highest hybridization intensities are identified as having hybridization intensities above an intensity threshold.

33. The computer program product of claim 29, wherein each nucleic acid probe includes n bases.

34. The computer program product of claim 33, further comprising:

code that compares bases at positions 1 through n of a first nucleic acid probe to bases at positions 1 through n of a second nucleic acid probe; and

code that counts base mismatches between the first and second nucleic acid probe.

35. The computer program product of claim 33, further comprising:

code that compares bases at positions 1 through n−

1 of a first nucleic acid probe to bases at positions 2 through n of a second nucleic acid probe; and

code that counts base mismatches between the first and second nucleic acid probe.

36. The computer program product of claim 29, wherein the code that sequences includes:

code that aligns the selected nucleic acid probes so that bases that are common overlap; and

code that sequences the nucleic acid sequence according to bases that occur most often at each position in the aligned nucleic acid probes.

37. The computer program product of claim 36, wherein the nucleic acid sequence is sequenced as complementary to the bases that occur most often.

38. A computer program product that sequences a nucleic acid sequence, comprising:

code that receives as input hybridization intensities for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence under conditions that do not allow identification of all nucleic acid probes of the plurality of nucleic acid probes that are perfectly complementary to part of the nucleic acid sequence;

code that selects nucleic acid probes from the plurality of nucleic acid probes, wherein the code that selects nucleic acid probes includes code that selects nucleic acid probes with highest numbers of single base mismatch neighbors among the probes, a single base mismatch neighbor being another probe that has the same sequence except for a single base that is different;

code that sequences and nucleic acid sequence according to the selected nucleic acid probes; and

a computer readable storage medium that stores the codes.

39. A computer program product that sequences a nucleic acid sequence, comprising:

code that receives as input hybridization intensities for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence under conditions that do not allow identification of all nucleic acid probes of the plurality of nucleic acid probes that are perfectly complementary to part of the nucleic acid sequence;

code that selects nucleic acid probes from the plurality of nucleic acid probes, wherein the code that selects nucleic acid probes includes code that selects nucleic acid probes that have fewer than a predetermined number of base mismatches with another probe;

code that sequences and nucleic acid sequence according to the selected nucleic acid probes; and

a computer readable storage medium that stores the codes.

40. A computer program product that sequences a nucleic acid sequence, comprising:

code that receives as input hybridization intensities for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence under conditions that do not allow identification of all nucleic acid probes of the plurality of nucleic acid probes that are perfectly complementary to part of the nucleic acid sequence;

code that selects nucleic acid probes from the plurality of nucleic acid probes, wherein the code that selects nucleic acid probes includes code that selects nucleic acid probes with highest hybridization intensities and the nucleic acid probes with the highest hybridization intensities are identified as having hybridization intensities above an intensity threshold;

code that sequences and nucleic acid sequence according to the selected nucleic acid probes; and

a computer readable storage medium that stores the codes.

41. A computer program product that sequences a nucleic acid sequence, comprising:

code that receives as input hybridization intensities for a plurality of nucleic acid probes, the nucleic acid probes hybridizing with the nucleic acid sequence under conditions that do not allow identification of all nucleic acid probes of the plurality of nucleic acid probes that are perfectly complementary to part of the nucleic acid sequence;

code that selects nucleic acid probes from the plurality of nucleic acid probes;

code that sequences and nucleic acid sequence according to the selected nucleic acid probes, wherein the code that sequences includes;

code that aligns the selected nucleic acid probes so that bases that are common overlap; and

code that sequences the nucleic acid sequence according to bases that occur most often at each position in the aligned nucleic acid probes; and

a computer readable storage medium that stores the codes.

42. The computer program product of claim 41, wherein the nucleic acid sequence is sequenced as complementary to the bases that occur most often.